Peer Review Report For: Bromelain Restores Glutamatergic Homeostasis via Regulation of NR2A, GLT-1, EAAC1, and xCT in Arsenic-Induced Cerebral Cortex and Hippocampal Neurotoxicity [version 2; peer review: 2 approved]

Background Chronic arsenic exposure interferes with hippocampal-dependent cognition through glutamate excitotoxicity, which in turn interferes with the regulation of receptors and transporters. Bromelain, a combination of proteolytic enzymes derived from Ananas comosus, is known to have neuroprotective effects; however, the mechanisms by which it counteracts glutamate-mediated toxicity in the brain are poorly understood. This study investigated the potential of bromelain to normalize glutamatergic homeostasis and cognitive function in arsenic-treated rats by specifically examining the NMDA receptor subunit NR2A and glutamate transporters GLT-1, EAAC1, and xCT. Methods Seventy-two adult male Wistar rats (200-220 g) were randomly divided into nine groups (n=8 each): control, arsenic-only (20 mg/kg/day sodium arsenite, administered via oral gavage for 14 days), bromelain-only (5, 10, or 15 mg/kg/day, oral gavage), arsenic plus bromelain (at the same doses), and arsenic plus donepezil (2 mg/kg/day, intraperitoneal). Sodium arsenite (20 mg/kg/day, p.o.) was administered for 14 days to induce neurotoxicity, followed by bromelain (5–15 mg/kg/day, p.o.) or donepezil (2 mg/kg/day) for an additional 14 days (post-exposure) treatment. Glutamate concentration was measured using sandwich Enzyme-Linked Immunosorbent Assay [ELISA] and total RNA was isolated to perform RT-qPCR to evaluate the expression of NR2A, GLT-1, EAAC1, and xCT. Results Arsenic exposure impaired spatial memory, increased brain tissue glutamate, and reduced NR2A and transporter gene expression. Post-exposure bromelain treatment partially/fully normalized these outcomes, with the most consistent effects at 10 mg/kg. Findings are based on mRNA expression and tissue glutamate. Conclusions Bromelain provides multi-target neuroprotection in arsenic neurotoxicity, rescuing glutamatergic homeostasis by coordinated upregulation of NMDA receptor NR2A and the transport network (GLT-1, EAAC1, xCT), accompanied by spatial learning and memory improvement. The demonstrated dose-response, showed numerically greater effects over 15 mg/kg donepezil, although direct superiority comparisons were not pre-specified. Also, the lack of overt adverse effects support its translational potential as a safe, plant-based adjunct to excitotoxic diseases and toxin-exposed populations.